The quantitative analysis of uric acid in a biological substance such as serum or plasma is a test which has to be carried out frequently and therefore plays an important part of clinical chemistry.
Previously, the enzymatic determination of uric acid has predominantly been carried out using the uricase method, which is used as a reference method, and by the method of Kageyama [Clin. Chim. Acta 31, 421 (1971)]. [See, also, Bergmeyer, "Methoden der enzymatischen Analyse", Volume 2, (1974), 1999-2005, Verlag Chemie)]. However, both methods have a number of disadvantages: In the uricase method, the high intrinsic absorbance of the serum sample at 293 nm is accompanied by a relatively low signal, due to the reduction in uric acid. Moreover, the procedure uses a wavelength for which the average laboratory does not have suitable equipment.
According to the Kageyama process, the H.sub.2 O.sub.2 formed by the action of the uricase is used for the oxidation of methanol to formaldehyde. The formaldehyde reacts with acetylacetone and ammonia, 3,5-diacetyl-1,4-dihydrolutidine being formed. However, at the end of the reaction a period of 60 minutes at elevated temperature (37.degree. C.) is required, and this is a major disadvantage when automatic analyzers are used.
Recently a process has become known [German Published Application (DE-OS) No. 24 50 726] wherein the H.sub.2 O.sub.2 formed is determined using catalase and aldehyde dehydrogenase, while NAD(P)H, which forms from NAD(P), can be measured photometrically at 334, 340, or 366 nm. This process has proven to be advantageous, as compared with processes used previously, particularly for determining uric acid content, since it takes only 15 minutes rather than 60 minutes, as in the past (Bergmeyer H. U., supra, pages 2002-2005). Consequently, this method is particularly suitable for automatic analyzers.
When this latest process was used, it was found that the serum contained constituents which deactivated the aldehyde dehydrogenase. Thus, it was found that after incubation of the serum with reagent and without uricase for 4 minutes and subsequent addition of uricase, the reaction is finished after about 10 minutes. If the serum is pre-incubated for 30 minutes, the reaction often does not come to an end until 20 to 30 minutes after the addition of uricase or else it continues as a creeping reaction (change in absorptions, .DELTA.A, per minute) lasting a considerable time. An additional problem is that the decrease in the aldehyde dehydrogenase activity occurs at different speeds in different sera.
This is a major disadvantage, particularly when the reagent is used in automatic analyzers. Longer pre-incubation periods are often required, because of the nature of the apparatus, before uricase is added as the starter reagent. Then, as a result of the reduction in the aldehyde dehydrogenase activity, the reaction may not be completed within the specified time.
The situation described gives rise to even greater problems in automatic analyzers which analyze substrates on a kinetic basis. Since it is the speed of the reaction which is being measured in such a case, the activity of the enzymes concerned must remain constant during the measuring period. If this is not the case, the reaction speed is affected not only by the quantity of substrate present but also by the fluctuating activity of the enzymes concerned, and is thus falsified. A constant aldehyde dehydrogenase activity must be ensured, particularly in those cases when the aldehyde dehydrogenase reaction is the rate-determining step of the reaction as a whole.
It is known from the literature [Black, S. Arch. Biochem. Biophys., 34 (1951), 86-97; Steinmann, C. R., and Jacoby, W. B., J. Biol. Chem., 242 (1967), 5019-5023] that aldehyde dehydrogenase is an enzyme containing thiol groups and that it can be stabilized with thiol reagents and/or with complex-forming agents. Ethylenediaminetetraacetic acid (EDTA), cysteine, glutathione, mercapto-ethanol, and thioglycolic acid are examples of reagents useful in this regard. EDTA is not sufficiently active as a protective reagent in the presence of serum. The other reagents mentioned certainly protect aldehyde dehydrogenase; however, in the incubation mixture their use results in a creeping reaction which, dependent upon the concentration of the substance added and on the serum being investigated, is from about 0.001 and 0.010 absorptions/minute. Representative values are set forth in the following table:
TABLE ______________________________________ Creeping Concentration reaction Additive (mol/liter) (.DELTA.A/min) ______________________________________ Dithiothreitol 5 .times. 10.sup.-4 0.002-0.003 Cysteine 5 .times. 10.sup.-4 0.005-0.007 Glutathione 5 .times. 10.sup.-4 0.004-0.007 Thioglycolic acid 5 .times. 10.sup.-4 0.001-0.002 ______________________________________